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Background Human papillomavirus (HPV)-associated cancers often exhibit resistance to immune checkpoint blockade (ICB) due to an immunosuppressive tumor microenvironment (TME), which is closely associated with TGF-β signaling. HPV oncoproteins, such as E6 and E7, promote TGF-β upregulation, which induces cancer-associated fibroblast (CAF) activation and collagen deposition, creating a physical and functional barrier to cytotoxic immune cell infiltration. Targeting both TGF-β and PD-L1 may overcome these immunosuppressive mechanisms and improve therapeutic outcomes.Methods We evaluated the therapeutic potential of YM101, a bispecific antibody simultaneously targeting TGF-β and PD-L1, in the TC-1 murine tumor model, which expresses HPV16 E6/E7 oncoproteins. In vivo tumor growth inhibition, survival benefit, and immune memory responses were assessed following treatment with YM101 or monotherapies. Multiplex immunohistochemistry and immunofluorescence were used to characterize spatial distributions of CD8 + T cells and collagen deposition. Flow cytometry profiled immune cell subsets within the TME, including cytotoxic T lymphocytes, NK cells, MDSCs, and macrophages. Bulk RNA sequencing was performed to evaluate transcriptomic changes and immune-related pathway enrichment. Public databases (TCGA, TIMER, GEPIA2) were used to explore the correlation between TGFB1, CAF markers (ACTA2, COL1A1), immune infiltration, and clinical outcomes across HPV-associated cancers.Results Anti-PD-L1 monotherapy had limited efficacy in the TC-1 model, with tumors displaying an immune-excluded phenotype characterized by extensive peritumoral collagen and low intratumoral CD8 + T cell infiltration. High expression of TGFB1, ACTA2, and COL1A1 correlated with poor prognosis and reduced immune infiltration in cervical cancer and HPV+ head and neck squamous cell carcinoma (HNSC), but not in HPV– HNSC. TGF-β blockade suppressed CAF activation and collagen production in vitro. YM101 treatment significantly suppressed tumor growth, prolonged survival, and induced complete regression in a subset of mice, with evidence of long-term immune memory. YM101 reshaped the TME by reducing TGF-β signaling and stromal fibrosis, promoting CD8+ T cell and NK cell infiltration, decreasing MDSCs, and shifting macrophage polarization toward an M1-like phenotype. Transcriptomic analysis revealed upregulation of immune activation and cytotoxicity-related genes (e.g., Ifng, Gzmb, Prf1) and enrichment of pathways related to T cell activation, cytokine signaling, and antigen presentation. YM101 also reduced tumor proliferation and enhanced apoptosis, as shown by reduced Ki67 and increased cleaved caspase-3 and TUNEL staining.Conclusions Dual blockade of TGF-β and PD-L1 with YM101 overcomes resistance to ICB in HPV-associated cancers by reversing TGF-β-mediated immunosuppression and reprogramming the TME. These findings support further clinical development of the humanized BsAb BiTP for patients with HPV-driven malignancies.